PROJECT SUMMARY/ABSTRACT 1,25-Dihydroxyvitamin D (1,25(OH)2D3) is synthesized in the kidney by Cyp27b1 and inactivated in all target tissues via Cyp24a1. The two genes are reciprocally regulated in the kidney by a number of hormones that serve to maintain appropriate physiologic levels of circulating 1,25(OH)2D3. In spite of their importance, however, little is known of the molecular details that facilitate expression of Cyp27b1 in the kidney or of Cyp24a1 in this tissue and elsewhere. This represents an enormous deficit in our understanding of the vitamin D system, a gap that is highlighted in a variety of diseases of mineral metabolism. In recent studies, we have identified key genomic regions in the Cyp27b1 locus in mice that mediate Cyp27b1 regulation by PTH, FGF23 and 1,25(OH)2D3, a module that is present only in the kidney. Deletion of components of this module reduce Cyp27b1 expression in the kidney but have no effect on Cyp27b1 expression in non-renal target cells (NRTCs). In view of these findings, we propose the following molecular studies. Aim 1: Characterize the endocrine module and its individual components in the mouse kidney that mediates Cyp27b1 expression and regulation by PTH, 1,25(OH)2D3 and FGF23 in vivo. We will use ChIP-seq analysis and CRISPR/Cas9 deletion studies in vivo to further characterize a regulatory module we have discovered in the kidney that functions to regulate the expression of Cyp27b1. We will also define the location of an independent module in the Cyp27b1 gene locus that mediates the actions of inflammatory signaling in NRTCs. These studies are designed to advance our understanding of the molecular mechanisms that underpin the metabolic activation of vitamin D. Aim 2: Characterize the regulatory sub-modules that control Cyp24a1 expression and regulation by PTH, 1,25(OH)2D3 and FGF23 in the kidney in vivo. Cyp24a1 in the kidney plays a coordinating role in regulating blood levels of 1,25(OH)2D3 and other vitamin D metabolites. We will use the methods outlined in Aim 1 to elucidate the genomic mechanisms through which this gene is downregulated by PTH and upregulated by both FGF23 and 1,25(OH)2D3. Mutational analysis in mice will allow us to define the clusters of enhancers and the sites of action of transcription factors and comodulators that are responsible for these activities. Aim 3: Utilize dietary manipulation to test and confirm regulatory hypotheses developed in Cyp27b1-compromised mouse strains relevant to disease. In this aim, we plan to test a hypothetical model that explains the complex phenotypes that have emerged following deletion of components of the kidney-specific regulatory module. We will use dietary manipulation of Ca, Pi, and vitamin D metabolites to test our hypotheses.